OAF is a DAF-like gene that controls ovule development in plants

We previously found that the RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1- (DAD1-) Activating Factor (DAF) controls anther dehiscence by activating the jasmonate biosynthetic pathway in Arabidopsis. Here, we show that in Arabidopsis, the DAF ancestor was duplicated into three genes (DAF, Ovule Activating Factor (OAF), DAFL2), which evolved divergent partial functions from their ancestor through subfunctionalization. In this case, DAF-DAD1-JA signaling regulates anther dehiscence, whereas OAF controls ovule development by negatively regulating cinnamyl alcohol dehydrogenase 9 (CAD9) activity and being negatively regulated by miR847 itself in Arabidopsis. Downregulation of OAF or upregulation of CAD9 and miR847 caused similar abortion of ovule formation due to precocious ovule lignification in transgenic Arabidopsis. Interestingly, only one DAF-like gene, PaOAF, exists in the monocot orchids, which has likely evolved through nonfunctionalization and maintains a conserved function as Arabidopsis OAF in regulating ovule development since defective ovules were observed in the virus-induced gene silencing (VIGS) PaOAF Phalaenopsis orchids. The absence of the DAF ortholog and its function in orchids is likely due to the evolution of stamens to a unique pollinium structure that lacks the feature of anther dehiscence. These findings expand the current knowledge underlying the multifunctional evolution and diverse functionalization of duplicate gene pairs within/among plants.

G ene duplication can generate additional new genes in the genome and is thought to be an important mechanism in evolution [1][2][3] . The majority of the duplicate gene pairs may retain overlap or have separated subsets of the original ancestral function (subfunctionalization) [3][4][5][6][7] . A low percentage of the duplicate gene pairs has been found to lose some copies due to genomic mutation (nonfunctionalization) 3,8,9 or to gain new functions that were not observed in the original ancestral gene (neofunctionalization) 1,4,10,11 . For example, it has been reported that~50% of the duplicated gene pairs in soybean (Glycine max L.) show subfunctionalization, whereas only 4% represent either non-or neofunctionalization 12,13 . Thus, duplicated genes tend to diverge in their expression and function, which is important for their retention in the genome of an organism, although the mechanism remains under investigation 2,3,14 . In addition, whether the same duplicate gene pairs evolved similarly in different plant lineages during evolution is interesting but remains poorly studied.
We have previously reported that the Arabidopsis RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1-(DAD1-) Activating Factor (DAF) specifically regulates anther dehiscence by activating the DAD1 gene in the jasmonate biosynthetic pathway 15 . There are two DAF-like genes (DAFL1 and DAFL2) that showed differential expression in flowers to DAF, were also identified in Arabidopsis 15 . DAFL1 is specifically expressed in ovules and carpel, whereas DAFL2 has an overlapping expression pattern with DAF in sepal/petal and with DAFL1 in carpel 15 . This suggests that DAF and DAFL1 may have evolved unique functions in regulating stamen and ovule development, respectively, during evolution. To uncover these questions, we comprehensively functionally characterized the DAFL1 gene and its regulatory networks and further explored the possible evolutionary diversification of the duplicate DAF-like gene pairs in Arabidopsis. Furthermore, we used virus-induced gene silencing (VIGS)-based gene knockdown to investigate the functional similarity/divergence of the Phalaenopsis orchid DAF-like gene to that in Arabidopsis.
Here, we show that Arabidopsis DAFL1, renamed Ovule Activating Factor (OAF), indeed has a distinct function from DAF in regulating ovule development, which is involved in upstream miR847 regulation and downstream CAD9 regulation. Thus, the expression divergence for duplicated DAF/OAF leads to functional divergence. In this study, we also found that only one DAFlike gene, PaOAF, was identified in Phalaenopsis orchids, whose expression pattern (in sepal/petal/anther cap/carpel/ovules) seems to be the sum of the three Arabidopsis DAF-like genes. Furthermore, Phalaenopsis PaOAF was found to be able to regulate ovule development through a mechanism similar to that of Arabidopsis OAF. Our findings provide a unique example to explore the multifunctional evolution of duplicated genes within/ among plants in which subfunctionalization likely occurred in Arabidopsis, whereas nonfunctionalization occurred in Phalaenopsis orchids for duplicate DAF-like gene pairs.

Results
Isolation of OAF cDNA from Arabidopsis. One Arabidopsis thaliana gene, Ovule Activating Factor (OAF) (At3g10910), was analyzed. This gene is known to be closely related to the E3 RING finger gene DEFECTIVE IN ANTHER DEHISCENCE1-(DAD1-) Activating Factor (DAF) (At5g05280) and DAFL2 (At5g01880), was previously named DAF-Like gene 1 (DAFL1), in the ATL subgroup 15 (Supplementary Fig. 1). OAF contains one exon without any introns and encodes a protein containing 181 amino acids that showed 58% and 47% identity to DAF and DAFL2, respectively ( Supplementary Fig. 2). Similar to DAF/DAFL2, a conserved C3H2C3-type RING finger motif 16,17 was identified in the C-terminus of the OAF protein, which showed 88/74% amino acid homology with those in DAF/DAFL2 ( Supplementary  Fig. 2). Furthermore, in the middle of the OAF protein, two other conserved motifs, a hydrophobic region and a GLD region, were also identified ( Supplementary Fig. 2).
OAF is a functional E3 ligase. His-SUMO-OAF-FLAG fusion proteins were produced by fusing SUMO to the N-termini of OAF through SUMO fusion technology 15,18 to further prove the role of OAF as an E3 ubiquitin ligase. In the presence of ubiquitin (Ub), E1 (UBA1) and E2 (UbcH5b), ubiquitination activity was detected with a polyclonal antibody against ubiquitin in the presence of purified His-SUMO-OAF-FLAG ( Supplementary  Fig. 3 . When the ubiquitination activity was analyzed for His SUMO-OAF(H135A)-FLAG and His-SUMO-OAF(H135/ 138A)-FLAG mutant proteins that were mutated in the RING finger domain and were unable to interact with E2 proteins, no ubiquitination signals were observed in the presence of either mutant protein ( Supplementary Fig. 3, Lanes 9-10). This further proved that an intact RING finger domain was required for the E3 ligase activity of OAF. This in vitro ubiquitination assay clearly proved that, similar to DAF 15 , OAF possesses E3 ligase activity and that this activity was completely abolished in the H135A and H135/138A mutants of OAF.
Detection of OAF expression by analysis of OAF::GUS Arabidopsis. To investigate the expression pattern of the OAF gene during flower development, OAF::GUS containing a β-glucuronidase (GUS) reporter gene driven by the OAF promoter was constructed, and GUS activity was analyzed in transgenic Arabidopsis. The results showed that GUS activity in OAF::GUS flowers was specifically detected in the carpel but not in the other three flower organs during all stages of flower development (Fig. 1a, b). The expression of GUS in the carpel was further analyzed. In OAF::GUS flower buds before stage 10, GUS was clearly detected in all tissues of the carpel, such as ovules, funiculus, transmitting tract, septum and pericarp (ovary wall) (Fig. 1a, c, d). Later, in OAF::GUS flowers after stage 12, GUS activity persisted in the funiculus, transmitting tract and septum and was completely absent in ovules and pericarp (Fig. 1b, e, f). This expression pattern was different from that observed in DAF::GUS flowers, in which GUS was mainly detected in the filaments and connective tissue of the anther throughout flower development 15 . The specific expression of OAF in ovules during the early stages of development revealed its possible role in controlling ovule development.
Ectopic suppression of OAF transcript or activity causes defects in anther dehiscence and ovule development in Arabidopsis plants. To further explore the role of OAF in regulating female reproductive development, antisense and RNAi strategies were applied to generate transgenic Arabidopsis plants in which the OAF gene and any putative functional redundant genes were repressed or silenced. The results indicated that a similar mutant phenotype with sterility was observed in both the 35S::OAF antisense ( Supplementary Fig. 4a, b) and RNAi ( Supplementary  Fig. 4g, h) plants. The siliques failed to elongate during late development ( Supplementary Fig. 4b, h), which was significantly different from the elongated and fully developed siliques in wildtype inflorescence ( Supplementary Fig. 4a, g). 35S::OAF antisense/ RNAi flowers opened normally and produced normal sepals, petals and carpels ( Supplementary Fig. 4e, i) similar to those observed in the wild-type flowers ( Supplementary Fig. 4c). Different from the normal dehiscence of the wild-type anthers after stage 12 of flower development ( Supplementary Fig. 4c, d), nondehiscent anthers were observed in these 35S::OAF antisense ( Supplementary Fig. 4e, f) and RNAi ( Supplementary Fig. 4i, j) flowers at all stages of flower development.
We further transformed 35S::OAF-H135A, a mutant form of OAF that contains a mutation in the RING motif of OAF (His-135 was substituted with alanine; Supplementary Fig. 5a), into Arabidopsis to generate dominant-negative, loss-of-function mutant plants for OAF. This strategy has been used successfully to generate dominant-negative mutants for several Arabidopsis RING-finger proteins, including the DAF we studied 15,[19][20][21] . Interestingly, short, undeveloped siliques ( Supplementary Fig. 5b, c) and indehiscence of the anther phenotype ( Supplementary Fig. 5d, e) were observed in these 35S::OAF-H135A plants, which were similar to those observed in the 35S::OAF antisense/RNAi flowers. As expected, OAF-H135A mRNA was upregulated in the 35S::OAF-H135A flowers ( Supplementary Fig. 5f).
The nondehiscent anthers produced in 35S::OAF antisense/ RNAi and 35S::OAF-H135A flowers were similar to those observed in 35S::DAF antisense/RNAi and 35S::DAF-dominantnegative mutant flowers 15 , indicating that OAF should be able to affect the function of DAF either by suppression of DAF expression (antisense/RNAi) or targeting the same substrate of DAF (dominant-negative mutant) once ectopically expressed in stamen/anther. This is supported by the result that not only the expression of OAF ( Supplementary Fig. 4k, n), but also DAF ( Supplementary Fig. 4l, o), was suppressed in 35S::OAF antisense ( Supplementary Fig. 4k, l) and RNAi ( Supplementary Fig. 4n, o) flowers. In addition, the expression of DAFL2, another closely related gene to OAF ( Supplementary Fig. 2), was also suppressed in 35S::OAF antisense ( Supplementary Fig. 4m) and RNAi ( Supplementary Fig. 4p) flowers.
Since the expression pattern suggests a possible role for OAF in controlling ovule development, testing whether ovule development was affected in 35S::OAF antisense/RNAi and 35S::OAF-H135A flowers was interesting. When the wild-type pollen grains were manually placed on the stigmas of 35S::OAF antisense/RNAi and 35S::OAF-H135A flowers, partial silique elongation and seed maturation were observed ( Supplementary Fig. 6c, e, g). Different from the full development of~40 seeds of an elongated wild-type silique ( Supplementary Fig. 6a, b, i), clear defective undeveloped ovules ( Supplementary Fig. 6d, f, h) and a reduction in seeds produced to~50% of that in wild-type were observed in the 35S::OAF antisense/RNAi and 35S::OAF-H135A siliques (Supplementary Fig. 6c, e, g, i). These results suggested that approximately half of the ovules from 35S::OAF antisense/RNAi and 35S::OAF-H135A flowers were defective and were unable to be fertilized with the wild-type pollen.
Specific expression of the dominant-negative H135A mutation for OAF in ovules causes defects only in ovule development in Arabidopsis. To confirm the function of OAF in specifically controlling ovule development, we further overexpressed OAF-H135A under its own promoter (pOAF::OAF-H135A) in transgenic Arabidopsis through the transactivation system 22,23 . The resulting plants showed a phenotype with partially elongated siliques during late development ( Fig. 1g-i). Different from that in 35S::OAF antisense/RNAi and 35S::OAF-H135A flowers, normal anther dehiscence with viable pollen grains released was observed in pOAF::OAF-H135A flowers (Fig. 1j-l), which was similar to that in wild-type flowers (Fig. 1m, n). When the partially elongated siliques were examined, defective and undeveloped ovules were clearly observed ( Fig. 1o-s), which caused añ 50% reduction in the seeds produced (Fig. 1t), similar to that observed in 35S::OAF-H135A x wild-type siliques ( Supplementary   Fig. 6g-i). This phenotype was correlated with the high expression level of OAF-H135A in the pOAF::OAF-H135A flowers (Fig. 1u). These results strongly support that OAF specifically controls ovule development during Arabidopsis flower development. The defects in anther dehiscence observed in 35S::OAF antisense/RNAi and 35S::OAF-H135A flowers were thus due to the ectopic expression of OAF antisense/RNAi or mutant proteins in anthers and sequentially altered the expression or activity of the duplicated gene DAF.
Identification of the OAF-interacting protein cinnamyl alcohol dehydrogenase 9 (CAD9). To seek the putative interacting or target proteins for OAF in regulating ovule development, a yeast two-hybrid (YTH) strategy using OAF as a bait to screen Arabidopsis flower bud cDNA library DNA was performed. Among the OAF-interacting proteins identified (Supplementary Table 1), a cinnamyl alcohol dehydrogenase 9 (CAD9) (At4g39330) gene specifically expressed in ovules was further characterized. CAD9 belongs to a CAD multigene family with nine genes in Arabidopsis ( Supplementary Fig. 7), which have been thought to be involved in lignin biosynthesis by catalyzing the last step of the monolignol biosynthetic pathway for the production of lignin monomers [24][25][26][27] . When CAD9::GUS transgenic Arabidopsis was generated, GUS activity was specifically and strongly detected in the ovules of the carpel during the early (before stage 10) (Fig. 2a, left) and late (after stage 12) (Fig. 2b-d) stages of flower development. The pattern of GUS expression indicated that CAD9 mRNA was strongly and constitutively expressed during all stages of ovule development.
To further test the stability of the CAD9 protein during different stages of ovule development, we generated a construct containing the GUS reporter gene fused to the CAD9 protein driven by a CAD9 promoter fragment (CAD9::CAD9 + GUS) and transformed it into Arabidopsis. Interestingly, GUS staining was significantly reduced in early developed ovules (stages 6-10) in these CAD9::CAD9 + GUS flowers (Fig. 2a, right) compared to the high GUS staining during the same stages of ovule and carpel development in CAD9::GUS flowers (Fig. 2a, left). In contrast, GUS staining was only slightly reduced in late developed ovules (stage 12) in these CAD9::CAD9 + GUS flowers (Fig. 2e, f) compared to GUS staining during the same stages of ovule development in CAD9::GUS flowers (Fig. 2b, c). These results c Close-up of the carpel from (a). O ovules, F funiculus, TT transmitting tract, P pericarp. Bar: 0.1 mm. d A cross-section of the carpel from (c). GUS was strongly detected in ovules (O) (arrowed), funiculus (F), transmitting tract (TT), septum (S) and pericarp (P). Bar = 50 µm. e Close-up of the carpel from (b). F funiculus, TT transmitting tract. Bar: 0.1 mm. f A cross-section of the carpel from (e). GUS was absent in ovules (O) (arrowed) and pericarp (P) and was only detected in funiculus (F), transmitting tract (TT) and septum (S). Bar = 50 µm. g An OAF::OAF-H135A (right) produced partially elongated siliques (red arrow), whereas wild-type plants (WT, left) produced long, well-developed siliques (white arrow). Bar: 2 cm. h Inflorescences from an OAF::OAF-H135A plant (right) with partially elongated siliques (red arrow) and inflorescence with elongated siliques (white arrow) from a wild-type plant (WT, left). Bar: 1 cm. i Close-up of the siliques from OAF::OAF-H135A (right) and wild-type plants (WT, left). j In OAF::OAF-H135A flowers, the anther was dehiscent and the pollen (arrow) was released after stage 12. Bar: 0.5 mm. k Close-up of the dehiscent anther and the pollen (arrow) from (j). Bar: 0.1 mm. Pollen grains with normal viability (stained dark red; arrowed) were observed in the OAF::OAF-H135A (l) and wild-type (WT) (m) anthers. Bar = 100 µm. n Close-up of the dehiscent anther and pollen (arrow) from wild-type (WT). Bar = 200 µm. o Defective and undeveloped ovules (arrowed) along with normal developing seeds (s) were produced from OAF::OAF-H135A partially elongated siliques. Bar: 0.1 mm. p Close-up of the defective and undeveloped ovules (o) from (o). f funiculus. Bar: 0.02 mm. q Defective and undeveloped ovules (arrowed) along with normally developed seeds (s) observed from mature OAF::OAF-H135A partially elongated siliques. Bar: 0.1 mm. r Close-up of the defective and undeveloped ovules (o) from (q). f funiculus. Bar: 0.02 mm. s An OAF::OAF-H135A partially elongated silique produced fewer than 20 seeds (arrowed). Bar: 1 mm. t Comparison of the total number of seeds produced in the OAF::OAF-H135A and wild-type (WT) siliques. Error bars show ± SEM. n = 10 and 20 biologically independent samples for wild-type and OAF::OAF-H135A, respectively. The asterisks "***" indicates significant difference from the wild-type (WT) value (***p ≤ 0.001). Statistical analysis was measured according to Student's t test. Box plots showed the median,10th, 25th, 75th and 90th percentiles as vertical boxes with error bars. u Analysis of the expression of OAF-H135A in the wild-type control (WT) and OAF::OAF-H135A plants. Error bars show ±SEM. n = 3 biologically independent samples. The asterisks "*" indicates significant difference from the wildtype (WT) value (*p ≤ 0.05). Statistical analysis was measured according to Student's t test.
indicated that CAD9 mRNA was constitutively expressed in all stages of ovule development. However, CAD9 proteins were unstable and constantly degraded during early ovule development and were stably maintained at a certain level during late ovule development.
CAD9 could be polyubiquitinated by OAF. To further prove the interaction between CAD9 and OAF and to demonstrate whether CAD9 is a substrate for OAF, His-SUMO-OAF-FLAG fusion proteins were used to detect the ubiquitination of the CAD9-HA-His. In the presence of ubiquitin (Ub), E1 (UBA1) and E2 (UbcH5b), ubiquitination activity was detected by Western blotting using anti-HA antibodies in the presence of purified His-SUMO-OAF-FLAG and CAD9-HA-His (Fig. 3, Lane 6 and Supplementary Fig. 8). In contrast, no ubiquitination activity was observed in the absence of E1, E2, E3 or Ub (Fig. 3, Lanes 1-5). For SUMO-OAF(H135A)-FLAG and SUMO-OAF(H135/138A)-FLAG mutant proteins that were mutated in the RING finger domain and were unable to interact with E2 proteins, this ubiquitination activity was absent (Fig. 3, Lanes 7-8). This in vitro ubiquitination assay clearly proved that OAF possesses E3 ligase activity and could interact and further polyubiquitinate CAD9 in the presence of E1, E2 and Ub. Ectopic expression of CAD9 causes defects in ovule development in Arabidopsis. Since our results for CAD9::GUS and CAD9::CAD9 + GUS revealed that the pattern of CAD9 protein levels was likely opposite to that of OAF during ovule development, where OAF was high in early and low in late stages, whereas CAD9 was low in early and high in late stages of ovule formation, CAD9 was suggested to be a possible target protein for OAF in regulating ovule development. To further confirm this assumption, CAD9 was ectopically expressed (35S::CAD9) in transgenic Arabidopsis. Similar to that in pOAF::OAF-H135A flowers, normal anther dehiscence with viable pollen grains released (Fig. 2h, i) and partially elongated siliques ( Fig. 2g and Supplementary Fig. 9a, b) with~50% defective and undeveloped ovules/seeds ( Fig. 2j-l) were observed in 35S::CAD9 flowers. This phenotype was correlated with the high expression level of CAD9 mRNA ( Supplementary Fig. 9c) in the 35S::CAD9 flowers. In addition, when the wild-type pollen grains were manually placed on the stigmas of 35S::CAD9 flowers, similar partial silique elongation ( Supplementary Fig. 9d, e) with~50% defective and undeveloped ovules was also observed (Fig. 2m, n). Furthermore, we ectopically expressed CAD9 under its own promoter (pCAD9::CAD9) in transgenic Arabidopsis through the transactivation system 22,23 . The resulting plants showed an identical phenotype to 35S::CAD9 plants by producing normal anther dehiscence with viable pollen grains released (Fig. 2p, q) and partially elongated siliques ( Fig. 2o and Supplementary Fig. 9f) with defective and undeveloped ovules (Fig. 2r, s), which caused an~50% reduction in the seeds produced (Fig. 2t). This phenotype was correlated with the high expression level of CAD9 mRNA (Fig. 2u) in the pCAD9::CAD9 flowers. These results strongly support that OAF specifically controls ovule development by negatively regulating CAD9 activity during Arabidopsis flower development. The defects in ovules were caused by a high level of CAD9 activity during early ovule development, which was either due to overexpression of CAD9 directly (35S::CAD9 and pCAD9::CAD9) or mutation of OAF function (pOAF::OAF-H135A), resulting in the activation of CAD9 activity.
35S::CAD9, pCAD9::CAD9, pOAF::OAF-H135A and 35S::OAF antisense/RNAi caused precocious lignification of the ovules. The next question is how exactly CAD9-OAF regulates ovule development. Since CAD family genes have been thought to function in lignin biosynthesis in plants [24][25][26][27] , we examined the status of ovule lignification in wild-type, 35S::CAD9, pCAD9::-CAD9, pOAF::OAF-H135A and 35S::OAF antisense/RNAi flowers. When the stage 10 flower carpel was treated with Mäule staining, which turns lignin a dark brown color, a significantly darker brown color was observed in the ovules of 35S::CAD9 (  GUS activity was specifically and strongly detected in the ovules (arrowed) of the carpel. s sepals, p petals, an anther, c carpel. Bar: 0.5 mm. c Close-up of the carpel from (b). O ovules, TT transmitting tract. Bar: 0.1 mm. d Close-up of the ovules (O) from (c). Bar: 0.1 mm. e A stage 12 flower of a CAD9::CAD9 + GUS transgenic plant. GUS activity was specifically detected in the ovules (arrowed) of the carpel and was weaker than that in CAD9::GUS in (b). s sepals, p petals, an anther, c carpel. Bar: 0.5 mm. f Close-up of the carpel from (e). O ovules. Bar: 0.1 mm. g A 35S::CAD9 plant (right) produced partially elongated siliques (red arrow in box), whereas wild-type plants (WT, left) produced long, well-developed siliques (white arrow). Bar: 2.5 cm. h In 35S::CAD9 flowers, the anther was dehiscent, and the pollen (arrowed) was released after stage 12. Bar: 0.5 mm. i Close-up of the dehiscent anther and the pollen (arrowed) from (h). Bar: 0.2 mm. j Defective and undeveloped ovules (arrowed) along with normal developed seeds (s) were produced from mature 35S::CAD9 partially elongated siliques. Bar: 0.1 mm. k Close-up of the defective and undeveloped ovules (o) from (j). f funiculus. Bar: 0.03 mm. l Comparison of the total number of seeds produced in the 35S::CAD9 and wild-type (WT) siliques. Error bars show ±SEM. n = 10 and 20 biologically independent samples for wild-type and 35S::CAD9, respectively. The asterisks "***" indicates significant difference from the wild-type (WT) value (***p ≤ 0.001). Statistical analysis was measured according to Student's t test. Box plots showed the median,10th, 25th, 75th and 90th percentiles as vertical boxes with error bars. m Defective and undeveloped ovules (arrowed) along with normal developed seeds (s) were produced from 35S::CAD9 x wild-type (WT)♂ partially elongated siliques. Bar: 0.1 mm. n Close-up of the defective and undeveloped ovules (o) from (m). f funiculus. Bar: 0.03 mm. o A CAD9::CAD9 plant (right) produced partially elongated siliques (red arrow in box), whereas wild-type plants (WT, left) produced long, welldeveloped siliques (white arrow). Bar: 2.5 cm. p In CAD9::CAD9 flowers, the anther was dehiscent, and the pollen (arrowed) was released after stage 12. Bar: 0.5 mm. q Close-up of the dehiscent anther and the pollen (arrowed) from (p). Bar: 0.1 mm. r Defective and undeveloped ovules (arrowed) along with normal developed seeds (s) were produced from CAD9::CAD9 partially elongated siliques. Bar: 0.1 mm. s Close-up of the defective and undeveloped ovules (o) from (r). f funiculus. Bar: 0.03 mm. t Comparison of the total number of seeds produced in the CAD9::CAD9 and wild-type (WT) siliques. Error bars show ±SEM. n = 10 and 20 biologically independent samples for wild-type and CAD9::CAD9, respectively. The asterisks "***" indicates significant difference from the wild-type (WT) value (***p ≤ 0.001). Statistical analysis was measured according to Student's t test. Box plots showed the median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars. u Analysis of the expression of CAD9 in wild-type control (WT) and CAD9::CAD9 plants. Error bars show ±SEM. n = 3 biologically independent samples. The asterisks "*" indicates significant difference from the wild-type (WT) value (*p ≤ 0.05). Statistical analysis was measured according to Student's t test. OAF is negatively regulated by its upstream 5'UTR during ovule development. It is interesting to note how OAF-CAD9 signaling is regulated. Based on the sequence analysis, an uORF (upstream open reading frame) containing 69 bp encoding 22 amino acids was found in the upstream 5'UTR of OAF gene 28 , named uORF for OAF (uOAF) (Supplementary Fig. 10). It has been reported that uORFs may regulate the transcription and translation of mORFs (major open reading frames) 29 . To determine the possible regulatory role of uOAF in OAF function, two constructs, 35S::F-OAF (containing both the uOAF + 5'UTR and OAF fragments) ( Supplementary Fig. 11a) and 35S::uOAF (containing the uOAF+5'UTR fragment only) ( Supplementary  Fig. 12a), were transformed into Arabidopsis, and the phenotypes were analyzed.
As expected, 35S::miR847 plants showed normal anther dehiscence (Fig. 4b, c) and partially elongated siliques (Fig. 4d, e), which contained defective ovules and exhibited an~50% reduction in the seeds produced (Fig. 4f-l), similar to that observed in pOAF::OAF-H135A mutant plants (Fig. 1o-t). This altered phenotype in 35S::miR847 plants was correlated with the high level of miR847 expression (Fig. 4m) and the downregulation of endogenous OAF and uOAF (Fig. 4n, o). Furthermore, precocious lignification of the ovules (Fig. 2v-10-9) was also observed in 35S::miR847 flowers. These results suggest that OAF expression was negatively regulated by miR847 through targeting its 5'UTR target sequences, which caused the degradation of OAF mRNA and resulted in an OAF knockdown phenotype.
Defects in ovule formation were observed in PaOAF-VIGS Phalaenopsis orchids. To further validate the function of the OAF ortholog, a Phalaenopsis orchid OAF (PaOAF) (Supplementary Fig. 1) was identified and characterized in this study. PaOAF encodes a protein of 192 amino acids that showed 62%/ 67% identity and 80%/83% similarity to OAF and DAF, respectively ( Supplementary Fig. 2). In Phalaenopsis flowers, PaOAF expression (Fig. 5e) was detected in the column and ovary of female reproductive organs (Fig. 5a-e), as well as in the anther  Bar: 0.2 cm. f Normally developing seeds (s) were produced from wild-type (WT) well-developed siliques. Bar: 0.1 mm. g Defective and undeveloped ovules (arrowed) along with normal developing seeds (s) were produced from 35S::miR847 partially elongated siliques. Bar: 0.1 mm. h Close-up of the defective and undeveloped ovules (o) from (g). f funiculus. Bar: 0.02 mm. i Defective and undeveloped ovules (arrowed) along with normally developed seeds (s) observed from mature 35S::miR847 partially elongated siliques. Bar: 0.2 mm. j Close-up of the defective and undeveloped ovules (o) from (i). f funiculus. Bar: 0.02 mm. k Normal seeds (arrowed) produced in wild-type (WT) well-developed siliques and two 35S::miR847 partially elongated siliques. Bar: 1 mm. l Comparison of the total number of seeds produced in the 35S::miR847 and wild-type (WT) siliques. Error bars show ±SEM. n = 10 and 20 biologically independent samples for wild-type and 35S::miR84, respectively. The asterisks "***" indicates significant difference from the wild-type (WT) value (***p ≤ 0.001). Statistical analysis was measured according to Student's t test. Box plots showed the median, 10th, 25th, 75th and 90th percentiles as vertical boxes with error bars. Analysis of the expression of miR847 (m), inOAF (n) and uOAF (o) in wild-type control (WT) and 35S::miR847 plants. Error bars show ±SEM. n = 3 biologically independent samples. The asterisks "**" and "***" indicates significant difference from the wild-type (WT) value (**p ≤ 0.01 and ***p ≤ 0.001). Statistical analysis was measured according to Student's t test.
cap, with complete absence in the pollinia (a coherent mass of pollen grains) of the male reproductive organ (Fig. 5c-e). In addition, PaOAF expression could also be detected in the sepals/ petals/lips (Fig. 5e). This finding revealed a possible similar function for orchid PaOAF and Arabidopsis OAF in regulating female reproductive organ development.
To pursue this question, virus-induced gene silencing (VIGS)based gene knockdown was performed to functionally analyze the PaOAF gene in Phalaenopsis orchids. In orchids, reproductive ovule development is triggered by pollination [30][31][32][33] . Fertilization occurred~70 days after pollination (DAP) once the ovule became mature in Phalaenopsis orchids 32 [33][34][35] . When the developing seeds were stained with Alexander's stain 36 130 DAP in control ovary, normal viability of the embryos (dark purple staining) and seed coats (purple staining) was observed (Fig. 5f, g). In contrast, PaOAF-VIGS ovaries contained defective seeds with abnormal viability of the embryos (clearly reduced size or complete absence) and defective seed coats (light green staining; Fig. 5h, i), which was correlated with the downregulation of PaOAF expression (Fig. 5j). Further analysis indicated that most PaOAF-VIGS seeds were defective and smaller than control seeds (Fig. 5k). In contrast to the observation that 80% of the control seeds germinated normally, more than 90% of PaOAF-VIGS seeds did not germinate (Fig. 5l, m). An interesting question is what caused the formation of defective ovules/embryos/seeds once PaOAF expression was suppressed. Since mutation in Arabidopsis OAF produced defective ovules due to the precocious lignification of the ovules, we examined the status of lignification for PaOAF-VIGS ovules 80 DAP. The results indicated that a clearly darker brown color for lignin staining was observed in the PaOAF-VIGS ovules (Fig. 5p, q) than in the control wild-type ovules (Fig. 5n, o). This result suggests that the defects in PaOAF-VIGS embryo/seed development were due to the precocious lignification of the ovules. Thus, the function and regulatory mechanism of OAF orthologs in controlling ovule development are conserved in the dicot Arabidopsis and monocot orchids.

Discussion
We previously found that a RING-type E3 ligase DEFECTIVE IN ANTHER DEHISCENCE1-(DAD1-) Activating Factor (DAF) controls anther dehiscence by activating the jasmonate biosynthetic pathway in Arabidopsis 15 . In this study, we further characterized a DAF-like gene, Ovule Activating Factor (OAF), which has a distinct function from DAF in regulating ovule development. In this OAF-ovule regulation network, to ensure that ovules are normally developed, OAF negatively regulates CAD9 activity by interacting and further ubiquitination of CAD9 and prevents early developing ovules from lignification, which harms and further limits the growth of ovules (Fig. 6). During the period from late-stage ovule development to embryo formation, the expression of OAF is negatively regulated by miR847, which specifically targets the 5'-UTR of OAF. The low activity of OAF causes the increased activity of CAD9, resulting in the formation of lignin in the inner integument and embryo sac to protect the further development of the embryos to mature seeds (Fig. 6). Thus, any alteration of OAF or CAD9 activity, for example, suppression of OAF or activation of CAD9 in early ovule development, will cause precocious lignification and defects in ovule development. The finding of the role of the miR847-OAF (E3 ligase)-CAD9 (target substrate) regulatory network in controlling ovule development by involving lignin formation provides a mechanism to explain ovule formation in plants. The role of OAF in regulating ovule development was further supported by the analysis of its ortholog PaOAF in Phalaenopsis orchids, since ovule development and further embryo/seed formation were defective in virus-induced gene silencing (VIGS) PaOAF knockdown Phalaenopsis orchids due to the precocious lignification of ovules (Fig. 6). This finding indicates the conserved function of OAF orthologs in plants.
The finding of the expression and functional divergence for putative duplicated genes DAF and OAF is interesting. Except in the pericarp of the carpel, DAF and OAF have no other overlapping expression pattern because DAF is expressed in sepal/ petal and filaments/connective tissue of the anther 15 , whereas OAF is expressed in the ovules/funiculus/transmitting tract/septum of the carpel 15 ( Fig. 1 and Supplementary Fig. 13). There is one more putative duplicated gene for DAF/OAF, named DAFL2, which is expressed in the sepal/petal and pericarp of the carpel 15 ( Supplementary Fig. 13). None of the three genes were expressed in pollen grains during stamen development. At least two gene duplication events would have occurred in Arabidopsis to generate DAF/OAF/DAFL2 (Fig. 6). Based on the phylogenetic analysis ( Supplementary Fig. 1), it is likely that the first event generated DAF/OAF and DAFL2 from the DAF/OAF-like ancestor, which have functions in regulating sepal/petal/anther/ carpel/ovule development, whereas the second event further generated DAF and OAF (Fig. 6). Mutation of DAF causes anther indehiscence, whereas mutation of OAF causes ovule defects. This observation suggests that their functions diverged during evolution, representing a case for the functional divergence between duplicated genes (Fig. 6). It is worth noting that although the expression and function of DAF and OAF have evolutionarily diverged in anthers and ovules, they may still share the same ability to bind substrates for each other. This is evidenced by the similar nondehiscent anthers produced in 35S::OAF-H135A ( Supplementary Fig. 5) and 35S::DAF-dominant-negative mutant flowers 15 , suggesting that OAF should be able to target the same substrate of DAF and affect the function of DAF once ectopically expressed in anthers. The overlapping expression pattern in sepal/petal for DAF/DAFL2 and the pericarp of the carpel for DAF/OAF/DAFL2 (Supplementary Fig. 13) may explain why no obviously altered phenotypes were seen in sepal/petal and Normal viability of the embryos with dark purple staining (red arrow) and seed coats with purple staining (black arrow) was observed. Bar = 100 µm. g Close-up of the developing seeds from (f), which showed normal developed embryos (red arrow) and seed coats (black arrow). Bar = 50 µm. h Alexander's staining of developing seeds 130 DAP in PaOAF-VIGS ovaries. Defective seeds with abnormal viability of the embryos that were either significantly reduced in size or completely absent (red arrow) were observed. Bar = 100 µm. i Close-up of the defective seeds from (h), which showed defective embryos (red arrow) and seed coats (black arrow). Bar = 50 µm. j Detection of PaOAF expression in control mock (C-1 to C-3) and PaOAF-VIGS Phalaenopsis V3 (V-1 to V-3) flowers by real-time quantitative RT-PCR. k Measurement of the size of the seeds from control mock and PaOAF-VIGS Phalaenopsis V3 ovaries. A total of 470 seeds from the control mock and 459 seeds from PaOAF-VIGS ovaries were used for measurement. l Normally germinated seedlings for the control mock (pCymMV) and the nongerminated seedlings from PaOAF-VIGS-deficient seeds. l leaf, r root. Bar carpel for DAF and OAF single mutations, respectively. However, whether DAFL2 regulates sepal/petal/carpel formation remains to be investigated (Fig. 6). Thus, based on the divergence of their expression and function, the three Arabidopsis DAF/OAF-like genes most likely experienced subfunctionalization after duplication. In this case, DAF retains the function in regulating sepal/ petal/anther/carpel development, OAF retains the function in regulating carpel/ovule development, while DAFL2 retains the function in regulating sepal/petal/carpel development from their DAF/OAF-like ancestor (Supplementary Fig. 13).
Unlike in Arabidopsis, only one DAF/OAF-like gene, PaOAF, was identified in Phalaenopsis orchids (Supplementary Fig. 1). This indicates that either no gene duplication or nonfunctionalization of duplicate genes occurred for the DAF/ OAF-like ancestor in orchids during evolution (Fig. 6). Why is only one PaOAF sufficient to perform the functions in orchids? We found that PaOAF is expressed in the sepal/petal, column/ ovary (female reproductive organs), and anther cap and is completely absent in pollinia (a coherent mass of pollen grains) ( Fig. 5e and Supplementary Fig. 13) in Phalaenopsis orchids. A similar expression pattern was also observed for PaOAF orthologs in Cymbidium and Cattleya orchids (Supplementary Figs. 14 and  15). Thus, the location for PaOAF expression in orchid flowers highly overlaps with that for DAF/OAF/DAFL1 in Arabidopsis flowers (Supplementary Fig. 13). This pattern reveals that PaOAF in orchids is able to perform most of the functions for DAF/OAF- Fig. 6 The multifunctional evolution of DAF/OAF-like genes in plants. In the eudicot Arabidopsis, the DAF/OAF ancestor duplicated into three genes (DAF, OAF, DAFL2) through two duplication events and eventually evolved to divergent functions through subfunctionalization in regulating various flower development. OAF retains its functions in regulating carpel/ovule development, DAF retains its functions in regulating sepal/petal/anther/carpel development and DAFL2 retains its functions in regulating sepal/petal/carpel development from their DAF/OAF-like ancestor. OAF regulates ovule development through a miR847-OAF-CAD9 signaling pathway. OAF negatively regulates CAD9 activity to prevent the early developing (before stage 10) ovules from lignification whereas miR847 acts upstream of OAF to suppress OAF expression during the period of late stage (after stage 12) ovule development, resulting in the increased activity of CAD9 to promote the formation of lignin in the ovules and ensure the normal embryo and seed development. In contrast to OAF, DAF retains the functions in specifically regulating anther dehiscence through a DAF-DAD1-JA signaling pathway and retains putative functions in regulating sepal/petal development similar to DAFL2 and carpel development similar to OAF/DAFL2 from their DAF/OAF-like ancestor. In the monocot orchid (Orchidaceae), only one PaOAF gene was identified which likely evolved to retain most of the DAF/OAF ancestor's functions. PaOAF maintains a conserved function similar to Arabidopsis OAF in regulating ovule development by preventing the early lignification of the developing ovules before 70 DAP. The decrease in PaOAF expression during late ovule development caused the formation of lignin and resulted in normal embryo and seed development. In OAF mutants and PaOAF-VIGS, precocious lignification of the ovules caused sequential defects in embryo/seed development. PaOAF may lose the function in regulating anther dehiscence as DAF due to evolutionary formation of the specific anther cap and pollinium structures in orchids. PaOAF may still have a function in regulating sepal/petal/carpel development in orchids, but this remains to be investigated.
like genes in Arabidopsis. The function of PaOAF in regulating ovule development in orchids was evidenced by the production of defective ovules/embryos/seeds in PaOAF-VIGS Phalaenopsis. Similarly, since no obviously altered phenotypes were observed in the sepals/petals/ovaries of PaOAF-VIGS flowers, whether PaOAF could regulate the formation of these organs remains to be investigated (Fig. 6).
The lack of function of PaOAF in male reproductive organs, such as the anther cap, can be explained. Orchids are highly evolved angiosperms whose stamens have evolved to a unique anther cap (related to anther structure) and a pollinium structure (Fig. 5c, d) that is tightly bound by pollen grains to facilitate insect pollination 37 . Different from plants such as Arabidopsis, there is no anther dehiscence or release of mature pollen in orchids. The mechanisms of pollinium determination remain obscure in orchids. Since the DAF function is to promote anther dehiscence in Arabidopsis, which is absent in the pollinium of orchids, there is no need for this function in orchids, and it is reasonably lost from PaOAF during evolution, even though the expression of PaOAF can still be detected in the anther cap.
In conclusion, a scenario (Fig. 6) was proposed to explain the evolutionary modifications of the functions of the DAF/OAF-like E3 ligase genes in plants. In the eudicot Arabidopsis, the DAF/OAF ancestor was duplicated into three genes (DAF, OAF, DAFL2) through two duplication events, which subsequently functionally diverged through subfunctionalization in regulating flower development. For example, miR847-OAF-CAD9 signaling regulates ovule development, whereas DAF-DAD1-JA signaling regulates anther dehiscence (Fig. 6). In the monocot orchid (Orchidaceae), only one PaOAF gene derived from a DAF/OAF ancestor exists, which retains the function of OAF in Arabidopsis in regulating ovule development but loses its function in regulating anther dehiscence. It is also possible that a newly duplicated DAF in plants such as Arabidopsis acquired an additional function in controlling anther dehiscence, which was originally absent in the DAF/OAF ancestor and PaOAF in orchids. These findings provide an excellent example of the diverse functionalization of duplicate gene pairs within/among plants and expand the current knowledge underlying the multifunctional evolution of duplicated genes in plants.

Methods
Plant materials and growth conditions. Arabidopsis seeds were germinated and grown as described previously 23,38,39 . Seeds for Arabidopsis were sterilized and placed on agar plates containing 1/2 X Murashige & Skoog medium 40 at 4°C for 2 days. The seedlings were grown in growth chambers under long-day conditions (16-h light/8-h dark) at 22°C for 10 days before being transplanted to soil. The light intensity of the growth chambers was 150 μE m −2 s −1 . Seeds for Phalaenopsis were germinated in BM-1 Terrestrial Orchid Medium medium (PhytoTechnology Laboratories®, United States) under long-day conditions (16-h light/8-h dark). Species and cultivars of orchids used in this study, including the moth orchids (Phalaenopsis Sogo Yukidian "V3"), Cymbidium spp., and Cattleya spp., were maintained in the greenhouse of National Chung-Hsing University, Taichung, Taiwan.
Construction of 35S::OAF antisense construct. cDNA containing the open reading frames of OAF (At3G10910) was amplified by RT-PCR using the 5' primer AtOAF-F and the 3' primer AtOAF-R for OAF. Both of the primers contained the generated KpnI recognition site (5'-GGTACC-3') to facilitate cloning of the cDNA. Sequences for the primers are listed in Supplementary Table 2. A KpnI fragment containing the full-length cDNA for the OAF gene was cloned into a binary vector pEpyon-32K (CHY Lab, Taichung, Taiwan) under the control of a cauliflower mosaic virus (CaMV) 35S promoter. The antisense constructs were orientationdetermined using PCR and were used for further plant transformation.
Construction of 35S::OAF RNA interference construct. For the 35S::OAF RNAi construct, a 312-bp DNA fragment containing a highly conserved sequence for OAF was obtained by PCR amplification using two primer pairs: AtOAF-RNAi-F1 (containing the XbaI recognition site 5'-TCTAGA-3') and AtOAF-RNAi-R1 (containing the PstI recognition site 5'-CTGCAG-3') for the sense fragment; and AtOAF-RNAi-R2 (containing the HindIII recognition site 5'-AAGCTT-3') and AtOAF-RNAi-F2 (containing the XhoI recognition site 5'-CTCGAG-3') for the antisense fragment. These two 312-bp XbaI-PstI and XhoI-HindIII fragments were subcloned into two sites of the intron-1 (500 bp) of Arabidopsis Actin-2 in the pBlueACTi vector (CHY Lab, Taichung, Taiwan), named OAF-i. A 1124 bp XbaI-XhoI fragment was then cut from OAF-i and subcloned into the binary vector pBI-mGFP1 (CHY Lab, Taichung, Taiwan) under the control of the CaMV 35S promoter, and it was then used for further plant transformation. The sequences of the primers are listed in Supplementary Table 2.
Construction of 35S::F-OAF and 35S::u-OAF constructs. A fragment (F-OAF) containing the cDNA of the open reading frame (ORF) and the 131 bp uOAF + 5'UTR for the OAF (Extended Data Fig. 10) was amplified by RT-PCR using the 5' primer AtFOAF-F and the 3' primer AtFOAF-R. A fragment (u-OAF) containing only the 150 bp uOAF + 5'UTR (Extended Data Fig. 11) for the OAF was amplified by RT-PCR using the 5' primer AtuOAF-F and the 3' primer AtuOAF-R. The primers contained the PstI (5'-CTGCAG-3') or SalI (5'-GTCGAC-3') recognition site to facilitate the cloning of the cDNA. The cDNA was cloned into a binary vector pEpyon-32K (CHY Lab, Taichung, Taiwan) under the control of a cauliflower mosaic virus (CaMV) 35S promoter and were used for further plant transformation. The sequences of the primers are listed in Supplementary Table 2.
Construction of 35S::miR847 construct. cDNA containing the open reading frames of miR847A precursor (At1G07051) was amplified by RT-PCR using the 5' primer AtmiR847-F and the 3' primer AtmiR847-R for miR847A. Mature sequence: UCACUCCUCUUCUUCUUGAUG. The primers contained the PstI (5'-CTGCAG-3') or SalI (5'-GTCGAC-3') recognition site to facilitate the cloning of the cDNA. The cDNA was cloned into a binary vector pEpyon-32K (CHY Lab, Taichung, Taiwan) under the control of a cauliflower mosaic virus (CaMV) 35S promoter and were used for further plant transformation. The sequences of the primers are listed in Supplementary Table 2.
Real-time PCR analysis. Total RNA was extracted 44,45 and used for quantitative real-time PCR which was conducted using a C1000 thermal cycler/Bio-Rad CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, California, USA) and Optical System Software version 3.0a (Bio-Rad Laboratories). For transcript measurements, the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd) was used. The amplification condition was: one cycle at 95°C for 1 min, followed by 40 cycles of 95°C (for 15 s), 58°C (for 15 s), and 72°C (for 15 s), and plate reading after each cycle. The name and the sequence of the gene-specific primers for Arabidopsis OAF, internal OAF (inOAF), uOAF, CAD9, miR847A, and for orchids (PaOAF of Phalaenopsis, CsOAF of Cymbidium and CaOAF of Cattleya) were listed in Supplementary Table 3. The data were analyzed using CFX Manager TM Software (Version 3.0; Bio-Rad Laboratories, Inc.). The transcript levels for genes were determined using three replicates and were normalized using reference housekeeping genes UBQ10 for Arabidopsis (AtUBQ10) 46,47 and ACTIN for orchids (PaACT4 for Phalaenopsis, OnACT for Cymbidium and Cattleya) 44,47 (Supplementary Table 3).  48 . Transformants were selected in a medium containing kanamycin (50 µg/ml) and were verified by RT-PCR analyses.
Lignin staining (Mäule staining). Lignin analysis was performed according to the method of histochemical staining of Arabidopsis thaliana secondary cell wall 51 . In Arabidopsis, the stage 10 and stage 14 flower samples were collected, and the floral organs (sepals, petals and anthers) were removed. Single valve was cut, removed from silique by needle and transferred to a 1.5 ml microcentrifuge tube. In Phalaenopsis Sogo Yukidian V3, cross-section of fruit 80 days after pollination was collected. One ml of the 0.5% potassium permanganate solution was added to the tube containing the samples. The tube was knocked gently and incubated for 2 min. In total, 750 µl of 0.5% potassium permanganate solution was replaced by adding 750 µl of distilled water to rinse out the potassium permanganate solution. This step was repeated until solution clear. The water solution was replaced by adding 1 ml of 3.7% HCl. After 2 min, the HCl solution was removed and 1 ml of concentrated ammonium hydroxide solution was added. Samples were put on glass slide, mounted with concentrated ammonium hydroxide solution and lignin was observed by microscope (olympus IX71).
Alexander's staining. For Arabidopsis pollen analysis, Alexander's staining solution 36 was added to cover the anthers cut from mature flowers and incubated at 37°C for 2 h. For orchid analysis, the developing seeds were mounted with Alexander's staining solution 36 and stand for 48 h at room temperature. The samples were observed under a microscope.
Virus-induced gene silencing (VIGS) experiment. The cDNA sequences of PaOAF were identified from Phalaenopsis V3 through Next-Generation Sequencing (NGS) analysis of Phalaenopsis floral buds with Arabidopsis OAF sequences. VIGS experiments on Phalaenopsis orchids were performed 44,47 and as described below. A DNA fragment in nonconserved regions of PaOAF was used for VIGS. DNA fragments were obtained by PCR amplification using the following primers (PaOAF-VIGS: VIGS-PaOAF-F and VIGS-PaOAF-R) and were inserted into the VIGS vector pCymMV-Gateway 44,47 independently. The attB sites (sequences in bold letters in Supplementary Table 2) were for in vitro recombination with attP sites in the VIGS vector pCymMV-Gateway to generate recombinant clones using Gateway ® BP Clonase II Enzyme Mix (Invitrogen™, Life Technologies, Carlsbad, CA, USA). pCymMV-Gateway-PaOAF and the empty pCymMV-Gateway as a control were transformed into Agrobacterium tumefaciens EHA105 for further inoculation. The leaf infiltration experiments in Phalaenopsis orchids 44,47 were performed as described below. Briefly, suspensions were injected into the leaf just above the site where the inflorescence emerged. At least three plants were inoculated with each pCymMV-Gateway construct for every infiltration. Flower buds and flower samples were collected and analyzed. For pollination experiments, blooming flower was pollinated and material was collected by 80 DAP (days after pollination) for lignin analysis, 130 DAP for embryo observation and 200 DAP for germination test.
Statistics and reproducibility. Data in the analysis of gene expression in various transgenic plants were analyzed using the two-sided Student's t test and represented as the mean ± SEM. In these cases, n = 3 biologically independent samples. The asterisks indicates significant difference from the wild-type (WT) value (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).
Reporting summary. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The data supporting the findings of this work are available within the paper, the Supplementary Information files and Supplementary Data 1 (including source data underlying main figures). Any other data sets generated and analyzed during this study are available from the corresponding author upon request.